Improved blood storage unit and method of storing blood

ABSTRACT

Blood storage units comprising containers with aqueous preservative solutions therein are improved by incorporating dihydroxyacetone (DHA) together with L-ascorbate (vitamin C) in the preservative solutions. The invention also relates to a method of storing human blood wherein viable red cells are stored in contact with both DHA and L-ascorbate. The preservative solution of the blood storage unit may also contain an anticoagulant such as citrate, a sugar energy source such as dextrose, and an ATP maintaining agent such as adenine. Where the blood storage unit is to be heat sterilized, as preferred, a blood bag providing a separate compartment for part of the preservative agents prevents deterioration of the preservative agents, the DHA and sugar energy source being heat sterilized separately for later admixture with the ascorbate, citrate anticoagulant and adenine. Separate pH control can also thereby be provided for the heat sterilization.

United States Patent [191 Deindoerfer et al.

[ Apr. 1, 1975 IMPROVED BLOOD STORAGE UNIT AND METHOD OF STORING BLOOD[75] Inventors: Fred H. Deindoerfer, Northridge;

Jon M. Brake, Burbank, both of Calif.

[73] Assignee: American Hospital Supply Corporation, Evanston, Ill.

[22] Filed: Mar. 29, 1973 [21] Appl. No.: 345,961

Related U.S. Application Data [63] Continuation-impart of Ser. Nos.194,652, Nov. 1, l97l, Pat. NO. 3,847,378, and Ser. No. 194,689, Nov. 1,1971, Pat. N0. 3,795,581.

[52 U.S. Cl 128/272, l95/1.8, 424/101 [51] Int. C13... A61J l/00, A61K17/00, C12K 9/00 [58] Field of Search 195/18; 424/101 [56] ReferencesCited UNITED STATES PATENTS 3,703,438 11/1972 Dovgalev 195/].8

Primary Examiner-Sam Rosen [57 7 ABSTRACT Blood storage units comprisingcontainers with aqueous preservative solutions therein are improved byincorporating dihydroxyacetone (DHA) together with L-ascorbate (vitaminC) in the preservative solutions. The invention also relates to a methodof storing human blood wherein viable red cells are stored in contactwith both DHA and L-ascorbate. The preservative solution of the bloodstorage unit may also contain an anticoagulant such as citrate, a sugarenergy source such as dextrose, and. an ATP maintaining agent such asadenine. Where the blood storage unit is to be heat sterilized, aspreferred, a blood bag providing a separate compartment for part of thepreservative agents prevents deterioration of the preservative agents,the DHA and sugar energy source being heat sterilized separately forlater admixture with the ascorbate, citrate anticoagulant and adenine.Separate pH control can also -thereby be provided for the heatsterilization.

15 Claims, 7 Drawing Figures SOLUTION B y 2 IMPROVED BLOOD STORAGE UNITAND METHOD OF STORING BLOOD CROSS-REFERENCES This application is acontinuation-inpart of our copending application Ser. Nos. 194.652 and194.689. both filed Nov. 1. 1971. now US Pat. Nos. 3.847.378 and3,795,581. respectively.

DRAWINGS The accompanying drawing. comprising FIGS. 1 to 7, illustratesone form of a blood collection and storage unit for use in practicingthe present invention where the preservative solutions are heatsterilized. The construction and method of use of such blood storageunits will be further described in Example IV.

BACKGROUND AND SUMMARY The state of the art with respect to biochemicalknowledge ofthe chemical makeup and functioning of red cells(erthyrocytes) is summarized in two recent publications: Red CellMetabolism and Function. edited by George J. Brewer. Plenum Press. 1970;and Red Cell Metabolism. Ernest Beutler. Grune and Stratton. 1971.

The principal energy source for red cells is glucose (or equivalentsugar) which is metabolized by the cells through complex biochemicalpathways involving enzymaticreactions. The principal pathway. oftenreferred to as the Embden-Meyerhoff pathway, involves the anaerobicbreakdown of glucose to pyruvic or lactic acid. An additional pathway isreferred to as the direct oxidative shunt or hexose monophosphate shunt.

In the Embden-Meyerhoff pathway, the compound l.3-diphosphoglycerate isproduced from D-glyceraldehyde-3-phosphate. The 1,3-diphosphoglycerateis converted by interaction with ADP (adenosine diphosphate) to ATP(adenosine triphosphate) and 3. phosphoglycerate, the reaction beingcatalyzed by phosphoglyceratc kinasc. An alternate by-path also leads to3-phosphoglycerate. by way of 2.3-diphosphoglycerate (hereinafterreferred to as 2,3-DPG or more concisely as DPG) an important regulatorof the oxygen affinity of hemoglobin. The complexing of 2.3- DPG withhemoglobin decreases the affinity of oxygen to hemoglobin in a manneressential to the release of oxygen to the body tissues.

In human blood. the normal level of 2,3-DPG is within the range from 12to 18 micromoles 2.3-DPG per gram of hemoglobin. Beutler gives a moreprecise figure: 15.36 i 1.98 micromoles 2.3-DPG/g. hemoglobin (Red CellMetabolism supra, p. 99). In the body, under usual conditions,sufficient 2,3-DPG is produced by the red cells in the metabolism ofglucose by the EmbdenMeyerhoff pathway to provide the required amountfor proper oxygen-hemoglobin-tissue transfer. For reasons that are notunderstood, however. the 2,3- DPG content of red cells in stored blooddecreases to subnormal levels interfering with oxygen released by thecells, even though blood is stored under refrigerator conditions (l-6C.) in admixture with an anticoagulant solution containing dextrose (orequivalent sugar) as the principal energy source for the red cells.Therefore. although the red cells remain viable. contain sufficient ATP,and provide a satisfactory survival rate (70 percent or more after 24hours), the subnormal 2,3-DPG content of the red cells may actuallycause a decrease in the oxygen delivered to the tissues for severalhours after the transfusion, and as long as 24 hours may be required forthe transfused red cells to be restored to normal 2.3-DPG levels foreffieent delivery of oxygen to the tissues. (Dawson. The HemoglobinFunction of Blood Stored at 4 C.." pp. 3()53l7, in Red Cell Metabolismand Function. supra).

The problem of administering stored blood deficient in 2.3-DPG isrendered more acute under many clinical conditions. such as patients inseptic shock. patients receiving large volumes of stored blood. andinfants. particularly premature infants. with infection or therespiratory disease syndrome. since the 2.3-DPG levels of the patientsblood may already be depressed. and further depression may occur onadministration of the low 2.3-DPG level blood.

Since the recognition of the function of 2.3-DPG as an oxygen releaseregulator for hemoglobin. and the recognition that depressed levels of2.3DPG can occur in the body and under in vitro storage of blood. therehas been a widespread search for chemical additives or other means ofmaintaining. or even increasing. the 2.3-DPG content of red cells. Ithas been found that frozen blood stored under very cold conditions (viz.C.) can be stored for many months without significant change in2.3-DPGlevels. However. because of the added expense in freezing blood andstoring it in the frozen conditions. the use of frozen blood has notbecome a commercial blood storage practice. The almost universalprocedure in the United States at the present time is to combine thefreshly collected blood with an anticoagulant solution containingdextrose, such as a citrate-dextrose solution or acitratephosphate-dextrose solution, and then to store the blood underrefrigeration at a substantially constant temperature within the rangefrom 1 to 6 C. Following this procedure, blood bank storage is approvedup to 21 days. and if the blood is not administered by that time. itusually'must be discarded.

In our copending applications, Ser. Nos. 194,652 and 194.689.cited-above. we have disclosed a blood storage unit and method of bloodstorage wherein dihydroxyacetone (DI-IA) is incorporated in thepreservative solution and maintained in contact with the red cells ofthe blood during storage for the purpose of maintaining and/orincreasing the 2.3-DPG content of the red cells. Subsequent to ourdiscovery of the effect of DHA on DPG levels of red cells, DR. ErnestBeutler. City of Hope Medical Center, Duarte, Calif. has found that themechanism of action of the DHA involves triokinase enzyme activity. atype of enzyme activity which had not previously been known to exist inred cells. By the postulated mechanism, dihydroxyacetone is converted todihydroxyacetone phosphate by the mediation of triokinase enzymeactivity. and the dihydroxyacetone phosphate enters the main metabolicpathway of the red cells.

Dr. Ernest Beutler has also reported that L-ascorbic acid (vitamin C)has a positive effect on the maintenance of DPG levels in stored blood,but that the mechanism of action of -L-ascorbate is unknown: TransfusionCongress, American Association of Blood Banks, XXV Annual Meeting, Aug.27-Sept. 2, 1972; and Western Society of Clinical Research, MleetingFeb. 3-5, 1972, Carmel. Calif. As reported by Dr. Beutler, red cellsstored with L-ascorbate use significantly less dextrose than controls,and the intracellular pH is significantly higher. Further, duringstorage of the red cells with ascorbate, less lactate and more pyruvateis formed from the sugar energy source. It therefore appears that themechanism iof action of ascorbate in maintaining DPG levels, althoughnot fully understood. is quite different from the mechanism of action ofDHA. Our discovery, which forms an important part of the presentinvention, was therefore unexpected: namely, that DHA and L-ascorbicacid (vitamin C) can function synergistically in maintaining and/orincreasing DPG levels of red cells in stored blood, especially where theblood is stored for 2 or 3 weeks or longer, such as storage periods offrom 3 to 6 weeks.

DETAILED DESCRIPTION In practicing the present invention, approved typesof blood collection and preservation containers are preferred. Eitherglass or plastic containers can be utilized, providing they meet theUSP. requirements. (See US. Pharmacopeia XVIII, pages 887 and 923.) Thecontainers will be sized for receiving and storing a predeterminedvolume of blood, such as 1000 ml., 500 ml., etc. Typically, thecontainers will have an internal volume adapted for receiving 500 ml. /2I.) of blood together with 70 to 125 ml. of anticoagulant solution. Inother words, the containers can have an internal volume of around 570 to625 ml. The containers will also be equipped with means for introducingthe fresh blood as it is collected, and for delivery of the blood intransfusion. Such transfusion and infusion assemblies used with theblood collection and storage units should meet U.S.P. requirements. (SeeUS. Pharmacopeia XVIII, p. 887).

As in the established practice, the anticoagulant solution for admixturewith blood collected in the containers should contain an anticoagulantsubstance to prevent coagulation of the blood, preferably, also. a sugarenergy source for the red cells in addition to the DHA. The preferredanticoagulant is citrate ions" which may be supplied by sodium citrate,or mixtures of citric acid and sodium citrate. The quantities to beemployed can be the same as in present practice (see U.S. pharmacopeiaXVIII, pages 47-49).

The sugar energy source for the red cells is preferably dextrose.However, it is known that other sugars are equivalent to dextrose forthis purpose, including fructose, mannose, and galactose. The amount ofdextrose or equivalent sugar employed can be the same as in presentpractice (see US. Pharmacopeia XVIII, pages 47-48). More specifically,from about 1.7 to 1.9 grams dextrose based on dextrose monohydrate canbe uti- 1 lized per 500 mililiters of blood.

In practicing the present invention, the blood collection andpreservation unit should contain at least and preferably at least 10,millimoles (mM) DHA per liter of blood. Consequently, when the unit isdesigned to collect 500 ml. of blood, at least 2.5 and preferably 5 mMof DHA will be incorporated in the aqueous anticoagulant solution. Whilethere does not appear to be any critical upper limit on the content ofDHA, there appears to be no reason to exceed I00 mM DHA per liter ofblood. When the container is designed for 500 ml. of blood, therefore,it will not be necessary to incorporate more than 50 mM of DHA in theanti-coagulant solution. Where the DHA is being utilized for 2,3-DPGmaintenance, and a sugar energy source is provided, as in presentpractice, it will usually not be necessary to employ more than 30 mM ofDHA per liter of blood, or 15 mM per 500 ml. of blood.

Since red cells occupy approximately one-third the volume of wholeblood, it will be appreciated that the red cells in storage will be incontact with an aqueous solution containing from 7.5 to mM of DHA perliter of solution, or preferably l5 to 45 mM DHA per liter of solution.Preferably. the DHA is incorporated in the blood immediately after itscollection.

For cooperation with the DHA in maintaining and/or increasing the DPGcontent of the red cells of the blood, the present invention utilizesL-ascorbic acid (vitamin C) as a cooperating additive. The L-ascorbicacid may be incorporated in the preservative solution in its free acidform, or as a water-soluble, bloodcompatible, non-toxic ascorbate salt.For example, the sodium salt of L-ascorbic acid can advantageously beused to obtain the same effect as adding ascorbate as free L-ascorbicacid. As used subsequently herein, therefore, the term L-ascorbate or,ascorbate is intended to refer to and include the L-ascorbate moietyboth as free acid and in salt form, either form being biologicallyequivalent for the purposes of the present invention.

On the basis of L-ascorbate content, the preservative solution foradmixture with the stored blood should provide 0.5 to 20 mM ofL-ascorbate per liter of blood. For example, where the preservativesolution is for admixture with substantially 0.5 liters of whole blood,from 0.25 to 10 mM L-ascorbate should be used in combination with 2.5 to50 mM Dl-IA. Preferably, from 1 to 10 mM of L-ascorbate per liter ofblood is employed. For example, when the preservative solution is to beadded to substantially 0.5 liters of blood, it can advantageouslycontain from 0.25 to 10 mM L- ascorbate together with 2.5 to 50 mM DHA.The red cells will therefore be stored in contact with an aqueoussolution containing from 0.75 to 30 mM L-ascorbate per liter ofsolution, or preferably from 1.5 to 15 mM L-ascorbate per liter ofsolution.

The DHA-ascorbate preservative solution also preferably containsadenine. For example, from 0.1 to 1.0 mM adenine can be incorporated inthe preservative solution per liter of predetermined blood volume. Inother words, where the preservative solution is for admixture withsubstantially O.5 liters of blood, the amount of adenine can range from0.05 to 0.5 mM.

For refrigeration storage, as described above, the conjoint action ofthe DHA-ascorbate combination of the present invention in maintainingDPG levels is accentuated as the length of the storage period increases.After storage of about 2 to 3 weeks, the synergistic cooperation of theL-ascorbate and the DHA becomes the predominant effect. With theDHA-ascorbate combination of the present invention, blood may be storedwhile maintaining acceptable DPG levels for periods of time over 3 weeksand up to 5 to 6 weeks. Data demonstrating the remarkable synergism ofDHA and L- ascorbate during the extended storage of blood is presentedbelow in Example I. Where adenine is incoporated in the preservativesolution, as preferred, the ATP (adenosine triphosphate) content of thered cells can also be maintained at a satisfactory level during suchextended storage periods.

The DHA-ascorbate combination of this invention can be utilized atpreservative pI-ls from neutrality (approximately pI-I 7.0) down to acidpI-Is as low as 5.0.

PHs on the acid side may be advantageous. For example. an admixture ofthe preservative solution with the blood. a pH in the range of 5.3 to5.9, such as a pH of substantially 5.6, is particularly advantageous.

Where the preservative solutions are sterilized by heat (autoclaving),as preferred. it has been discovered that the decomposition of the DHAand the ascorbate can be minimized by dividing the preservative solutioninto two separate solutions for purposes of sterilization, the solutionsbeing recombinable for admixture with the blood within the bloodcollection container. Specifically it has been discovered that ascorbatewhen heat sterilized tends to be decomposed by DHA and also by dextrose.Consequently, it is preferred to provide the blood storage unit with aseparate compartment containing an aqueous solution of DHA and dextrose,the blood bag, or other compartment, containing an aqueous solution ofthe ascorbate. The DHAdextrose aqueous solution component has been foundto be most sta ble when heat sterilized at a pH within the range from3.8 to 4.2, such as a pH of substantially 4.0. This pH is thereforepreferred. The ascorbate containing solution component canadvantageously have a pH of 5.3 to 5 .9, such as a pH of substantially5.6. This component can also contain the citrate anticoagulant and theadenine, all of these ingredients being substantially stable under heatsterilization in admixture with each other under the stated pH.Alternatively, however, all ingredients of the preservative solution canbe combined, and the aqueous solution can be sterilized by passing itthrough a sterilization filter before being filled into the bloodstorage container. This procedure, however, is more difficult andexpensive than heat sterilization.

Various aspects of the present invention are further illustrated by thespecific examples set out below:

EXAMPLE I This example describes actual laboratory experi ments andreports the data obtained, which demonstrate the synergistic effect ofdihydroxyacetone (DHA) and L-ascorbic acid (vitamin C) on2,3-diphosphoglycerate (DPG) in stored blood. In three separateexperiments, blood from a single donor was divided into four portions.One was stored with CPD-adenine,

one with CPD-adenine-ascorbate, one with CPD- adenine-DHA, and one withCPD-adenine-ascorbate- DHA. The concentrations of the components were asfollows: CPD-adenine, CPD (citrate-phosphatedextrose) per U.S.P. XVlll,pg. 48-49, and adenine, 0.5 mM per liter of blood; L-ascorbic acid (L-ascorbate), 100 mg. per each 100 ml. blood; and dihydroxyacetone (DHA),20 mM per liter blood. The pH of the preservative solution was 5.6.

Samples were stored in 100 ml. plastic blood bags at 4 C. and sampled atintervals. DPG was determined by the enzymatic method of Prins and Loos,as described in Red Cell Metabolism and Function, ed. G. J. Brewer, pp.227-288 (Plenum Press, 1970).

In Experiment N0. 1, blood was drawn into heparin (2115 U.S.P. units/500ml. blood). Forty ml. of blood were transferred to the sterile 100 ml.plastic bags containing 6 ml. of CPD-adenine. 1n Experiments 2 and 3,blood was drawn in CPD-adenine m]. anticoagulant/500 ml. of blood).Aliquots of the blood were then transferred aseptically to sterile ml.plastic bags.

A 10% solution of DHA was prepared and sterilized by autoclaving. It wasadded to selected bags in a ratio of 0.5 ml. per 500 ml of blood. Asolution of L- ascorbate was prepared by dissolving 5 grams of L-ascorbic acid in 100 ml. of water and adjusting to pH 5.5 with l Nsodium hydroxide. It was sterilized by filtering through a 0.22 micronsterilizing filter, and was added to selected bags in a ratio of l ml.per 50 ml. of blood.

The results of these experiments are shown in Table A. After 3 weeks ofstorage, the synergism of DHA and ascorbate on DPG levels is revealed.For this purpose, synergism can be measured when the DPG level of theDHA plus ascorbate sample exceeds the sum of the DPG level of ascorbatealone plus DHA alone. At 3 weeks, such synergism was measured in one ofthree experiments. At 4 weeks and 5 weeks, the synergism was measured in2 out of 3 experiments. At 6 weeks, synergism was measured in all 3experiments.

In Table B, the same data are recalculated as difference values, sampleminus control. This isolates the effect on DPG due to the additive fromthe effect due to TABLE A Effect of Ascorbate. DHA and the Combinationof Ascorbate/DHA on DPG Levels of Blood Stored in CPD-Adenine StorageTime DPG (9? of initial) Additive (weeks) Exp. No. 1 Exp. No. 2 Exp. No.3 Average None 3 12 12 22 15.3 Ascorbate 3 18 66 71 51 .7 DHA 3 67 71 9577.7 DHA Ascorbate 3 137* 109 150 132* None 4 10 15 14 13 Ascorbate 4 1471 60 48.3 DHA 4 41 26 25 30.7 DHA Ascorbate 4 1 19* 86 125* None 5 l3 815 12 Ascorbate 5 25 39 56 40 DHA 5 28 1O 20 19.3 DHA Ascorbate 5 125*48 96* None 6 13 1 1 19 14.3 Ascorbate 6 32 13 50 31.7 DHA 6 5 8 20 1 1DHA Ascorbate 6 88* 36* 84* 693* *Sum of DPG value for ascorbate and DHAalone is less than DPG value: for ascorbate and DHA together.

TABLE B Effect of Ascorbatc. DHA and the Combination of Ascorhatc /DHAon Differential DPG Levels of Blood Stored in CPD-Adenmc um ol' DPG\alucs of ascorhate and DHA separately is less than DPG \aluc of thecombination of the two. Calculated as the difference in DPG between thesample and the control CPD-adenine.

1e CPD-adenine preservative. The synergism is even lore clearlyevidenced in these results; namely syner- .stic action is disclosed in 2out of 3 experiments at 3 'eeks, and in 3 out of 3 experiments at 4, 5,and 6 eeks. It is therefore apparent that synergistic cooperaon of DHAand ascorbate in maintaining DPG levels I stored blood provides a meansfor greatly improving 1e quality of the blood.

EXAMPLE 11 In one embodiment, the invention may be practiced follows:

To prepare a CPD-adenine-ascorbate- .hydroxyacetone system, dissolve thefollowing chemals in 800 ml of water for injection U.S.P. and add aterto make one liter of solution: sodium citrate dihyate 30.8 grams (g),dextrose (anhydrous) 22.2 g. dildroxyacetone 14.7 g, adenine 0.55 g,L-ascorbic acid 1 g, and sodium biphosphate monohydrate 2.22 g. ierilizeby filtration through a 0.22 micron sterilizing ter. Using aseptictechnique fill 70 milliliters (ml) to sterile blood bags of volumecapacity for collection 500 ml of blood. Pack the prepared units in,metal ms under nitrogen until needed for blood collection 1d storageuse. Where the blood in admixture with the DHA and vcorbate is storedfor periods beyond 1 week. as prerred, it is desirable to invert thestorage containers at ast at the end of each week of storage. In oneprerred procedure, the storage containers are inverted lily, or at least5 days per week. Such inversion serves provide a mild agitation of thecontents of the blood 1g. thereby maintaining the red cells in moreuniform mtact with the solution of DHA and ascorbate. This ill help toassure that the combined effects of the HA and the ascorbate aremaximized.

EXAMPLE [11 will be subsequently described in detail in Example IV. Ingeneral, the unit consists ofa 500 ml. blood bag with a 15 ml. pilottube attached. The solution for the blood bag (Solution A) is preparedby dissolving the following in 800 ml. of water and adding water to makeone liter final volume: sodium citrate dihydrate 38.3 g., adenine 0.68g., ascorbic acid 11.1 g., and sodium biphosphate monohydrate 2.76 g.

With the pilot tube clamped off, 56.4 ml. of this solution is filledthrough a donor tube into the blood bag. Then cyclohexane is applied tothe end of the donor tube, and it is inserted into a needle adaptor withattached needle. This seals the needle to the tubing.

The solution for the pilot tube (Solution B) is prepared by dissolvingthe following in 800 ml. of water and bringing to a final volume of oneliter: dextrose (anhydrous) 105.6 g. and dihydroxyacetone (66.9 g.). ThepH is adjusted to 4 by adding 1 N sodium hydroxide. The separatecompartment provided by the pilot tube is connected at its inner end tothe blood bag, by a releasably clamped tubing. Then 15.4 ml. of thissolution is added to the pilot tube, through the short filling tubingconnected to the outer end of the pilot tube. This filling is then heatsealed.

The bag unit can be used as follows: After opening the can, the bag isremoved and the clamp between the pilot tube and the bag is opened. Thepilot tube is squeezed, forcing Solution B into the main bag. The clampon the pilot line is closed, and the bag is agitated to mix Solutions Aand B thoroughly. The needle protector is removed, and a venipuncturewas made by the usual technique in a human volunterr. After 500 ml. (530g.) of blood is collected, the clamp on the donor line is closed. Thebag is stored on its side in a 4 C. refrigerator, and agitated toresuspend the red cells in the plasma, as described in Example 11.

EXAMPLE IV In the accompanying drawings, there is shown a blood storageunit which is adapted for the practice of the present invention. Thefigures of this drawing are related as follows:

FIG. I is an elevational view of a complete blood storage unit ready forthe collection of blood;

FIG. 2 is a perspective view of one of the two clamps of the unit ofFIG. 1;

FIG. 3 is an exploded elevational view of the needle adaptor and needlecover of the unit of FIG. 1;

FIG. 4 is a detailed view showing the clamped portion of one of thetubes of FIG. 1;

FIG. 5 illustrates the appearance of the clamped portion of the tube ofFIG. 4 immediately after the removal of the clamp;

FIG. 6 illustrates the appearance of the clamped portion of the tube ofFIG. 4 after the clamp has been removed and the tube opened for the flowof liquid; and

FIG. 7 is a sectional view taken on line 77 of FIG. 5 showing the tubein collapsed condition as it would appear when clamped or before openingthe tube for liquid flow.

As referred to in Example III, the blood storage unit includes astandard flexible plastic blood bag 10 having a blood storagecompartment 11 therein, and a pilot tube 12 providing a separate smallerliquid storage compartment 13 therein. As indicated on FIG. 1,compartment 11 contains Solution A while compartment 13 containsSolution B. It will be understood that these solutions may be preparedand incorporated in these compartments as described in Example Ill.

Although the constructional details of the blood bag unit of FIG. I areconventional and well known in the blood collection and storage art,they will be briefly described in order that the use of the bloodstorage unit for the purpose of the present invention may be clearlyunderstood. Blood bag 10 which may be formed by a heat sealing procedurefrom a suitable plastic sheet material such as polyvinylchloride isprovided with an inlet 14 connected to an inlet tube 15. As illustrated,tube 15, which may be longer than illustrated ifdesired, connects to aYconnector 16. From the Y-connector there extends a blood collectiontube 17 having a needle assembly 18 at the outer end thereof and a lineclamp 19 thereon adjacent a slidable sleeve 20. As shown more clearly inFIG. 3, the needle assembly 18 includes a hub 19, a needle and a needlecover or protector 21. From connector 16 there also extends a tube 22which connects to the inner end of the enlarged pilot tube 12 and withthe compartment 13 therein. On tube 22, there is also provided a lineclamp 19 and an adjacent sleeve 20. It will be understood that the tube17 and 22 may be longer than shown if desired. At the other end of thepilot tube 20, compartment 13 connects to a short filling tube 23.

As indicated in Example III, Solution A will be filled into compartment11 through tube 17 before the needle assembly 18 is attached to theouter end thereof, the clamp 19 on line 17 being open during thisfilling operation, while the clamp 19 on line 22 is closed. Followingthe filling of Solution A through tube 17, clamp 19 can be moved toclosed position and needle assembly 18 attached. As shown more clearlyin FIG. 2, clamp 19 provides an enlarged opening 19a through which thetubing can extend without being clamped, and this opening communicateswith the restricted slot 19b within which the tubing is clamped to atemporarily sealed condition.

Also, as indicated in Example III. Solution B is introduced into theseparate compartment 13 through the filler tube 23 with the clamp 19 online 22 in closed position. After the filling operation. the filler tube23 may be heat sealed as indicated at 24. During heat sterilizationwhich may be carried out as described in Example III, the clamps 19 onlines 17 and 22 may remain closed. For collection of blood, theprotector 21 will be removed from the needle 20, clamp 19 opened and thetube held in oepn condition by means of sleeve 20. The blood from thedonor will then be transferred through lines 17. and 15 to thecompartment 11. After the blood has been collected, clamp 19 on line 17may again be moved to closed position. Either prior to the collection ofthe blood or subsequent thereto, Solution B may be mixed with Solution Aand with the blood in compartment 11 by opening clamp 19 and movingsleeve 20 to hold tube 22 in open condition. Since the pilot tube 20 isformed of a flexible plastic material. it can be squeezed to provide apump action forcing Solution B through tubes 22 and 15 into compartment1]. Tube 12 may also be elevated to assist this transfer by gravityflow. After the transfer of Solution B to compartment 11, the clamp 19on line 22 may again be moved to closed position. Where it is desired tomake the unit more compact for storage of the collected blood, and afterthe blood and Solution B are both in compartment 11, the tubeils'may beheat sealed, as indicated at 25 and then clipped off, as indicated at26.

The procedure for manipulating the clamp 19 and the sleeve 20 inrelation to a tube T, such as the tubes 17 or 22 of FIG. 1, isillustrated in FIGS. 4 to 7. In FIG. 4, clamp 19 is shown in its raisedor clamping position, the tube T being squeezed to a temporarily sealedcondition by its engagement in the slot 19b. to open the tube, clamp 19is moved in relation to tube T so that the tube extends through thelarger opening 19a, and is then moved away from the previously clampedportion by sliding it down the tube. As shown in FIGS. 5 and 7, theclamped portion 27 of the tube T tends to remain sealed after removal ofthe clamp 19. It can be opened by squeezing it between a thumb andforefinger. After opening, the sleeve 20 is pushed over the previouslyclamped portion of the tube to hold the tube in open condition. Thisposition is illustrated by FIG. 6. Since such use and manipulation ofsuch clamps and sleeves are well known in the blood collection andadministration art, it is not believed to be necessary to furtherdescribe them herein.

Conveniently, all of the components of the blood collection and storageunits of FIG. 1 can be formed of suitable plastic materials. Forexample, bag 10, pilot tube 12, tubes 15, 17, 22 and 23 and Y connector16 may be formed of polyvinyl chloride, slide clamps 19 of nylon orother relatively rigid thermoplastic, and hub 19 and protector 21 ofpolyvinyl chloride or other suitable thermoplastic. Needle 21 ispreferably formed of stainless steel of a standard needle size, such asa l6 gauge needle.

It will be understood, as shown, that bag 10 is provided with thestandard hanging loops and perforations, for example, as indicated at 28and 29. The top of the bag is also provided with a pair of tubularconnector outlets 30 having their outer ends closed by tear-off caps 31.For administration of the blood to a patient, one of the caps 31 can beremoved, and a blood administration set connected to one of the tubes30.

It will be apparent to those skilled in the art that the bloodcollection unit of FIG. 1 can be modified in various ways while stillbeing usable for the practice of the present invention. For example, thepilot tube 12 may be replaced by a small separate bag, or bag can bemanufactured with two compartments, and means provided for opening aseal between the two compartments to mix Solutions A and B aftercompletion of the heat sterilization.

EXAMPLE V This example describes laboratory experiments demonstratingthat DHA and ascorbate can be added to blood after one week of storage,resulting in the maintenance of high DPG levels for 6 weeks. Fivehundred ml. of human blood were collected in a blood bag containing 70ml. of CPD-adenine (composition given in Example 1). Four 35-ml.aliquots of the blood were transferred to sterile 100 ml. blood bags,one bag serving as a control and the others being used in otherexperiments. The bags were stored at 4 C. for 1 week, and then DHAmM/l.) and L-ascorbate (5.7 mM/l.) were added to one bag as follows: asterile injection site (a spike with a rubber septum attached) wasplaced in one of the ports of the blood bag. Then using a sterilesyringe, the following solutions were injected into the large blood bag:3.8 ml. of a 2 molar solution of DHA, sterilized by autoclaving at 250F. for 10 minutes, and 7.6 ml. of a 5 percent solution of L- ascorbicacid adjusted to pH 5.6 with sodium hydroxide and sterilized byfiltration through a 0.22 micron sterile filter. All bags were mixeddaily except weekends.

The results of DPG assays of the blood are shown in Table C. The controlshowed a rapid fall in DPG levels after the first week, while the bloodsupplemented with DHA and ascorbate at 1 week of storage maintainednormal or higher than normal DPG levels for 6 weeks.

TABLE C DPG Levels in Blood Collected in CPD-Adenine With and WithoutAddition of DHA/Ascorbate After One Week of Storage at 4 C.

DPG (mM/g Hb) Storage Time C PD-ad C PDad+DHA+ascorbate For practicingthe method described in Example V, the blood can be collected in anystandard blood storage bag or container, and at the time of collection,mixed with a standard anti-coagulant containing citrate ions and a sugarenergy source such as dextrose. For example, the CPD anti-coagulantdescribed in Example I can be employed, and, if desired, adenine mayalso be included, as described in Example I. The container should beprovided with means for subsequently introducing an aqueous solution ofdihydroxyacetone and L-ascorbate. For example, a solution for additionto 0.5 liters of blood can be prepared by dissolving 0.90 grams of DHAand 0.44 grams of L-ascorbic acid in ml. of

water, and then subjecting the solution to sterile filtration.

We claim:

1. A blood storage unit comprising a container for receiving and storinga predetermined volume of blood and preservative solution admixable withthe blood stored in said container, said preservative solution beingsterile and providing a sugar energy source and an anti-coagulant forpreserving said blood, said preservative solution also providing forcooperative admixture with said stored blood an amount ofdihydroxyacetone (DHA) equal to 5 to millimoles (mM) per liter of saidpredetermined blood volume together with an amount of L-ascorbate equalto 0.5 to 20 mM per liter of said predetermined blood volume.

2. The blood storage unit of claim 1 wherein said DHA is present in anamount of from 10 to 30 mM of DHA per liter of said predetermined bloodvolume.

3. The blood storage unit of claim 1 wherein said preservative solutionalso provides adenine in an amount equal to from 0.1 to 1.0 mM per literof said predetermined blood volume.

4. The blood storage unit of claim 1 wherein said preservative solutionprovides from 1 to 10 mM of said L- ascorbate per liter of saidpredetermined blood volume.

5. The method of maintaining the 2,3-diphosphoglycerate (2,3-DPG)content of viable red cells of whole human blood, comprisingincorporating in said whole blood from 5 to 100 millimoles (mM) ofdihydroxyacetone (DHA) per liter of said blood together with 0.5 to 20mM of L-ascorbate per liter of said blood, and holding said blood withsaid red cells in contact with said DHA and L-ascorbate for sufficienttime to maintain their 2,3-DPG content at a level resulting from thesynergistic action of said DHA and said L-ascorbate.

6. The method of claim 5 wherein said DHA and said L-ascorbate areincorporated in said blood in amounts of from 15 to 45 mM DHA and 1.5 to15 mM ascorbate per liter of blood. I

7. The method of maintaining 2,3-diphosphoglycerate (2,3-DPG) content ofthe red cells of whole human blood under storage conditions, comprisingadding to said whole blood from 5 to 100 millimoles (mM) ofdihydroxyacetone (DHA) and from 0.5 to 20 mM of L- ascorbate per literof blood, and storing said DHA and ascorbate containing blood withoutfreezing at a temperature below 10 C.

8. The method of claim 7 wherein from 10 to 30 mM of said DHA and from 1to 10 mM of said L-ascorbate are added to said blood immediately afterthe collection thereof.

9. The method of claim 7 in which said blood is stored for a period offrom 3 to 6 weeks.

10. A preservative solution for addition to stored blood, comprising asterile aqueous solution of dihydroxyacetone (DHA) and L-ascorbate, saidsolution containing from 0.5 to 20 mM of'said L-ascorbate per each 5 to100 mM of said DHA.

11. A preservative solution for addition to substantially 0.5 liters ofwhole blood, comprising a sterile aqueous solution containing from 2.5to 50 mM dihydroxyacetone together with 0.25 to 10 mM of L- ascrobate.

12. A heat-sterilized blood storage unit, comprising a container forreceiving and storing a predetermined volume of blood, a first sterileaqueous preservative solution in said container, means providing aseparate compartment, a second sterile aqueous preservative solution insaid compartment, means permitting said second solution to be introducedinto said container for admixture with said first solution and with saidpredetermined volume of blood. said first and second solusource and saidDHA being contained only in the other of said first and secondsolutions.

13. The blood storage unit of claim 12 wherein said DHA is present in anamount of from 10 to 30 mM of DHA per liter of said predetermined bloodvolume.

14. The blood storage unit of claim 12 wherein one of said preservativesolutions also contains adenine in an amount equal to from 0.1 to 1.0 mMper liter of said predetermined blood volume.

15. The improved blood storage unit of claim 13 wherein said onepreservative solution contains from I to 10 mM of said L-ascorbate perliter of said predetermined blood volume.

1. A BLOOD STORAGE UNIT COMPRISING A CONTAINER FOR RECEIVING AND STORINGA PREDETERMINED VOLUME OF BLOOD AND PRESERVATIVE SOLUTION ADMIXABLE WITHTHE BLOOD STORED IN SAID CONTAINER, SAID PRESERVATIVE SOLUTION BEINGSTERILE AND PROVIDING A SUGAR ENERGY SOURCE AND AN ANTI-COAGULANT FORPRESERVING SAID BLOOD, SAID PRESERVATIVE SOLUTION ALSO PROVIDING FORCOOPERATIVE ADMIXTURE WITH SAID STORED BLOOD AN AMOUNT OFDIHYDROXYACETONE (DHA) EQUAL TO 5 TO 100 MILLIMOLES (MM) PER LITER OFSAID PREDETERMINED BLOOD VOLUME TOGETHER WITH AN AMOUNT OF L-ASCORBATEEQUAL TO 0.5 TO 20 MM PER LITER OF SAID PREDETERMINED BLOOD VOLUME. 2.The blood storage unit of claim 1 wherein said DHA is present in anamount of from 10 to 30 mM of DHA per liter of said predetermined bloodvolume.
 3. The blood storage unit of claim 1 wherein said preservativesolution also provides adenine in an amount equal to from 0.1 to 1.0 mMper liter of said predetermined blood volume.
 4. The blood storage unitof claim 1 wherein said preservative solution provides from 1 to 10 mMof said L-ascorbate per liter of said predetermined blood volume.
 5. Themethod of maintaining the 2,3-diphosphoglycerate (2,3-DPG) content ofviable red cells of whole human blood, comprising incorporating in saidwhole blood from 5 to 100 millimoles (mM) of dihydroxyacetone (DHA) perliter of said blood together with 0.5 to 20 mM of L-ascorbate per literof said blood, and holding said blood with said red cells in contactwith said DHA and L-ascorbate for sufficient time to maintain their2,3-DPG content at a level resulting from the synergistic action of saidDHA and said L-ascorbate.
 6. The method of claim 5 wherein said DHA andsaid L-ascorbate are incorporated in said blood in amounts of from 15 to45 mM DHA and 1.5 to 15 mM ascorbate per liter of blood.
 7. The methodof maintaining 2,3-diphosphoglycerate (2,3-DPG) content of the red cellsof whole human blood under storage condiTions, comprising adding to saidwhole blood from 5 to 100 millimoles (mM) of dihydroxyacetone (DHA) andfrom 0.5 to 20 mM of L-ascorbate per liter of blood, and storing saidDHA and ascorbate containing blood without freezing at a temperaturebelow 10* C.
 8. The method of claim 7 wherein from 10 to 30 mM of saidDHA and from 1 to 10 mM of said L-ascorbate are added to said bloodimmediately after the collection thereof.
 9. The method of claim 7 inwhich said blood is stored for a period of from 3 to 6 weeks.
 10. APRESERVATIVE SOLUTION FOR ADDITION TO STORED BLOOD, COMPRISING A STERILEAQUEOUS SOLUTION OF DIHYDROXYACETONE (DHA) AND L-ASCORBATE, SAIDSOLUTION CONTAINING FROM 0.5 TO 20 MM OF SAID L-ASCORBATE PER EACH 5 TO100 MM OF SAID DHA.
 11. A preservative solution for addition tosubstantially 0.5 liters of whole blood, comprising a sterile aqueoussolution containing from 2.5 to 50 mM dihydroxyacetone together with0.25 to 10 mM of L-ascrobate.
 12. A HEAT-STERILIZED BLOOD STORAGE UNIT,COMPRISING A CONTAINER FOR RECEIVING AND STORING A PREDETERMINED VOLUMEOF BLOOD, A FIRST STERILE AQUEOUS PRESERVATIVE SOLUTION IN SAIDCONTAINER, MEANS PROVIDING A SEPARATE COMPARTMENT, A SECOND STERILEAQUEOUS PRESERVATIVE SOLUTION IN SAID COMPARTMENT, MEANS PERMITTING SAIDSECOND SOLUTION TO BE INTRODUCED INTO SAID CONTAINER FOR ADMIXTURE WITHSAID FIRST SOLUTION AND WITH SAID PREDETERMINED VOLUME OF BLOOD, SAIDFIRST AND SECOND SOLUTIONS TOGETHER PROVIDING A SUGAR ENERGY SOURCE, ANANTICOAGULANT, AN AMOUNT OF DIHYDROXYACETONE (DHA) EQUAL TO 5 TO 100MILLIMOLES (MM) PER LITER OF SAID PREDETERMINED BLOOD VOLUME, AND ANAMOUNT OF L-ASCORBATE EQUAL TO 0.5 TO 20 MM PER LITER OF SAIDPREDETERMINED BLOOD VOLUME, SAID L-ASCORBATE BEING CONTAINED ONLY IN ONEOF SAID FIRST AND SECOND SOLUTIONS PRIOR TO SAID ADMIXTURE THEREOF, ANDSAID SUGAR ENERGY SOURCE AND SAID DHA BEING CONTAINED ONLY IN THE OTHEROF SAID FIRST AND SECOND SOLUTIONS.
 13. The blood storage unit of claim12 wherein said DHA is present in an amount of from 10 to 30 mM of DHAper liter of said predetermined blood volume.
 14. The blood storage unitof claim 12 wherein one of said preservative solutions also containsadenine in an amount equal to from 0.1 to 1.0 mM per liter of saidpredetermined blood volume.
 15. The improved blood storage unit of claim13 wherein said one preservative solution contains from 1 to 10 mM ofsaid L-ascorbate per liter of said predetermined blood volume.